Most conventional pharmaceuticals currently in use for the treatment of serious disorders such as cancer and inflammatory diseases do not selectively accumulate at the site of disease [Bosslet et al., 58, 1195-1201 Cancer Res. (1998)]. For example, intravenously administered drugs distribute evenly within the different organs and tissues of the body, rather than selectively accumulating at the site of disease.
One approach to circumvent the disadvantages of conventional pharmacological therapies involves the preferential delivery of a bioactive agent to the site of disease by means of a binding molecule specific for a pathology-associated marker [Neri & Bicknell (2005) Nature Rev. Cancer, 4, 436-446]. The selective targeting of the drug to the diseased tissue will ultimately result in an increased local concentration at its site of action, sparing normal organs from the unwanted effects of the bioactive agent used to confer a pharmacological benefit (e.g., a growth factor, an enzyme, a hormone, an anti-inflammatory drug, a cytotoxic drug, a cytokine, a radionuclide, a photosensitizer). In most cases, this will lead to an improved therapeutic index of the delivered pharmaceutical, i.e. a higher efficacy with minimized side effects. Indeed, the favourable toxicity profile of site-specific therapeutics may open new avenues in the therapy of angiogenesis-related diseases, allowing the systemic administration of highly potent and promising agents, which are currently either given at suboptimal doses or whose clinical application has to date been impeded by unacceptable side-effects when applied in an unmodified form.
Ligand-based pharmacodelivery strategies fundamentally rely on the identification of good-quality markers of pathology, allowing a clear-cut discrimination between diseased tissues and healthy organs. Monoclonal antibodies and their fragments represent the preferred agents for pharmacodelivery applications [Rybak et al. 2, 22-40 Chem. Med. Chem (2007); Shrama et al., 5, 147-159 Nat. Rev. Drug Discovery (2006)], but globular protein mutants [Binz and Plückthun, 23, 1257-1268 Nature Biotechnology (2005)], peptides [Sergeeva et al., 58, 1622-1654, Adv. Drug. Deliv. Rev. (2006)] and even small organic ligands [Low et al., 41, 120-129, Acc. Chem. Res. (2008)] are also increasingly being used.
Antibody-based targeted delivery of bioactive agents to sites of angiogenesis as a therapeutic strategy for cancer treatment has been described. In the case of inflammatory disorders, antibody-based targeted delivery is much less well studied. The applicant has previously demonstrated that the ED-A domain of fibronectin, and the ED-B domain of fibronectin, two marker of angiogenesis, are expressed in the arthritic paws in the collagen-induced mouse model of rheumatoid arthritis. Using both radioactive and fluorescent techniques, the human monoclonal antibody F8, specific to ED-A, and the human monoclonal antibody L19, specific to ED-B, were found to selectively localize at sites of inflammation in vivo, following intravenous administration. When such antibodies were fused to the anti-inflammatory cytokine interleukin-10 the conjugate strong therapeutic activity was also shown (PCT/EP2007/004044, PCT/EP2008/009070). Nevertheless there remains a need in the art for further antibodies which can be employed in ligand-based pharmacodelivery applications for the treatment and diagnosis of diseases, such as cancer and inflammatory disorders.
IIICS Isoform of Fibronectin
Fibronectins (FN) are multifunctional, high molecular weight glycoprotein constituents of both the extracellular matrix and body fluids. They are involved in many different biological processes such as the establishment and maintenance of normal cell morphology, cell migration, haemostasis and thrombosis, wound healing and oncogenic transformation [Alitalo et al., (1982) Adv Cancer Res, 37 111-158; Yamada, (1983) Curr Opin Cell Biol, 1, 956-963; Hynes, (1985) Annu Rev Cell Biol, 1, 67-90; Ruoslahti et al., (1988) Annu Rev Biochem, 57, 375-413; Owens et al., (1986) Oxf Sury Eukaryot Gene, 3, 141-160]. Structural diversity in FNs is brought about by alternative splicing of three regions (ED-A, ED-B and IIICS) of the primary FN transcript (Hynes, R., (1985) Annu Rev Cell Biol, 1, 67-90; Zardi et al., (1987) EMBO J, 6, 2337-2342) to generate at least 20 different isoforms, some of which are differentially expressed in tumour and normal tissue. For example, five different splice isoforms of the human IIICS isoform of fibronectin have been described. As well as being regulated in a tissue- and developmentally specific manner, it is known that the splicing pattern of FN-pre-mRNA is deregulated in transformed cells and in malignancies (Castellani et al., (1986) J Cell Biol, 103, 1671-1677; Borsi et al., (1987) J Cell Biol, 104, 595-600; Vartio et al., (1987) J Cell Sci 88, 419-430, Zardi et al., (1987) EMBO J, 6, 2337-2342; Barone et al., (1989) EMBO J, 8, 1079-1085; Carnemolla et al., (1989) FEBS Letter 215, 269-273; Oyama et al., (1989) Biochemistry, 28, 1428-1433; Borsi et al., (1992) Exp Cell Res 199, 98-105). The FN isoforms containing the ED-A, ED-B and IIICS sequences are expressed to a greater extent in transformed and malignant tumour cells than in normal cells.
Much of the information relating to the expression of the IIICS isoform of fibronectin in healthy and diseased tissues derives either from mRNA studies or from studies with monoclonal antibodies (antibodies FDC-6 and X18A4). These antibodies were generated by hybridoma technology following immunization with fibronectin and immunosuppression with cyclophosphamide. Antibody FDC-6 binds to a specific O-linked N-acetygalactosaminylated hexapeptide epitope within the fibronectin type III connecting segment (IIICS) [Matsuura et al., (1985) PNAS, 82, 6517-6521; Matsuura et al., (1988) J Biol Chem, 263, 3314-3322]. However, since the antibody requires both the peptide backbone and the carbohydrate moiety to recognize the epitope, it is not suitable for targeting application especially when cross-reactivity between species is needed. Antibody X18A4 recognizes a different IIICS region than FDC-6, but the binding epitope has never been fully characterized [Feinberg R. et al., (1995) Am J Obstet Gynecol, 172, 1526-1536]: the main application for antibody X18A4 is related to the detection of oncofetal fibronectin in the cervix of pregnant women to predict preterm labour. There is evidence that IIICS expression is modulated in rheumatoid arthritis and osteoarthritis: in particular, it seems that the isoform 89V (CS1) is up-regulated in inflammation [Kriegsmann J et al., (2004) Rheumatol Int, 24, 25-33; Elices M J et al., (1994) J Clin Invest, 93, 405-416].
Matrix-metalloproteinase 3 (MMP3)
Matrix metalloproteinase 3 (also known as stromelysin 1) is a member of a family of more than 20 zinc-dependent extracellular enzymes with a key role in tissue remodeling [Nagase and Woessner, (1999) J Biol Chem, 274, 21491-21494; Martin and Matrisian, (2007) Cancer Metastasis Rev, 26, 717-724; Vartak and Gemeinhart, (2007) J Drug Target 15(1) 1-20]. Abnormal expression of various MMP proteins has been shown to play a role in a variety of disease types including cancer progression and in inflammatory conditions such as rheumatoid arthritis [Martin and Matrisian, (2007) Cancer Metastasis Rev, 26, 717-724; Brinckerhoff and Matrisian, (2002) Nat Rev Mol Cell Biol, 3, 207-214; Overall and Kleifeld, (2006) Nat Rev Cancer, 6, 227-239]. The catalytic domain of MMP3 is known to be relatively well conserved between mouse, rat and man.
Periostin
Periostin or Osteoblast Specific Factor 2 (OSF-2) was described for the first time in 1999 by the group of A. Kudo as a dimeric protein of 90 kDa secreted by osteoblasts and osteoblast-like cell lines. Periostin is mainly localized in the extracellular matrix of the periosteum and its main function is to act as a cell adhesion molecule for preosteoblasts and to induce osteoblast attachment and spreading [Horiuchi K et al., (1999) J Bone Miner Res, 14, 1239-1249].
The periostin N-terminal region contains four fasciclin-like domains (FAS1-4) as well as several glycosylation sites. Six different splice isoforms of periostin have been reported, but only four of them have been sequenced and annotated: these splice isoforms are characterized by the presence or absence of casette exons 17 to 21 at the C-terminus of the protein [Castronovo et al., (2006) Mol Cell Proteomics, 5, 2083-2091; Litvin et al., (2004) J Cell Biochem, 95, 1044-1061; Kim et al., (2008) Int J Oncol, 32, 161-169].
Tai and colleagues produced a monoclonal anti-periostin antibody by hybridoma technology and detected, by Western blotting, expression of the human periostin protein in the adrenal glands, lung, thyroid, uterus, vagina, ovary, testis, prostate, and in the gastrointestinal tract, with a preferential expression in the stomach and colorectum, while lower levels were noted in the small intestine and esophagus (Tai et al., Carcinogenesis, 26, 908-15, 2005). There have been observations of periostin being associated with a number of disorders. For instance, Morra and Moch [Virchows Archives., (2011) 459, 465-475] described the role of periostin in tumor microenviroment and tumor development. The N-terminal domain, with the FAS domains, bind to integrins and activates the Akt/PKB and the FAK-mediated signaling pathways, thus leading to tumor invasion and metastasis. The C-terminus of the protein binds to ECM molecules and influences the organization of the ECM. It is not yet clear if the different splice isoforms play different roles in ECM modification in tumor invasion.
In an in vivo chemical proteomic analysis, based on the terminal perfusion of three mouse models of liver metastases, periostin was one of the most abundant accessible antigens identified [Borgia et al., (2010) Cancer Res, 70, 309-318]. Immunofluorescence analysis confirmed the proteomic findings, indicating that periostin can be expressed in the neovasculature of tissues undergoing extensive remodeling.
Tenascin-W
Tenascin-W is the most recently discovered member of the tenascin gene family. Human tenascin-W is composed of a modular structure shared with all other tenascins (i.e. tenascin-C, -R, and -X respectively), which includes: N-terminal heptad repeats, 3.5 EGF-like repeats, 9 FN III domains and a C-terminal fibrinogen-related domain. In contrast to other tenascin family members, the existence of tenascin-W splice isoforms has not been reported to date [Chiquet-Ehrismann et al., (2011) Cold Spring Harb Perspect Biol. 3 (5), doi: 10.1101/cshperspect.a004960].
During embryonic development, tenascin-W is predominantly expressed in the extracellular matrix of periosteal bone, and to a lesser extent in smooth muscle, tendons and ligaments [Weber et al., (1998) J Neurobiol, 35, 1-16; Scherberich et al., (2004) J Cell Sci, 117, 571-581; Meloty-Kapella et al., (2006) Dev Dyn, 235, 1532-1542]. In healthy adults, the expression of tenascin-W is strongly reduced and restricted to the kidney, cardiac valves, corneal limbus and periosteum [Scherberich et al., (2004) J Cell Sci, 117, 571-581]. Tenascin-W expression has been detected in the perichondrium/periosteum during endochondral ossification and bone fracture repair [Kimura et al., (2007) Biochem Biophys Res Commum, 356, 935-941], suggesting its association with osteogenesis [Martina et al., (2010) Int J Biochem Cell Biol, 42, 1412-1415].
Tenascin-W is a tumour-associated antigen; its expression partly overlaps with tenascin-C in various cancers. To date tenascin-W expression has been detected in the stroma of breast [Degen et al., (2007) Cancer Res, 67, 9169-9179], and colorectal cancer [Degen et al., (2008) Int J Cancer, 122, 2454-2461], gliomas [Martina et al., (2010) FASEB J, 24, 778-787], melanomas, as well as pancreatic, kidney, and lung carcinomas [Brellier et al., (2012) BMC Clin Pathol, 4, 12-14]. Tenascin-W was not detectable in the corresponding healthy tissues. Interestingly, in most tumor types, tenascin-W was detected in the perivascular region of newly formed blood vessels [Brellier et al., (2012) BMC Clin Pathol, 4, 12-14, Martina et al., (2010) FASEB J, 24, 778-787].
The amino acid sequence of tenascin-W is highly conserved, between human, mouse and rat. However two major differences are present. Firstly, the three tenascin-W orthologues differ in the number of FNIII domains, with the mouse and rat variants containing 3 additional FNIII domains. Secondly, the mouse and rat tenascin-W genes contain a putative integrin binding RGD motif located in the second FNIII domain, which is absent in the human orthologue.